The present invention relates generally to a device for separating molecules on the basis of their size. More particularly, the present invention relates to a device for separating molecules on the basis of their size where the device is adapted to work in a multitude of centrifugation devices and allows for the separation of molecules in a very small sample size.
In the course of experiments, assays, or analytical measurements, it is often necessary or desirable to separate components in a mixture. Such separations may be accomplished by a variety of methods, including, but not limited to, salt precipitation, solvent precipitation, adsorption, and chromatography. The method of separation that is chosen is often selected with reference to the characteristics and stability of the molecular species being separated.
When it is desired to separate molecular species of different sizes, gel filtration chromatography, also known to those familiar with the art as size exclusion chromatography, is the method often chosen. Technically, gel filtration chromatography refers to the separation of components in an aqueous environment, while size exclusion chromatography refers to the separation of components in a non-aqueous environment. However, it is understood to those familiar with the art that these terms are considered interchangeable and may be used synonymously. Commercial chromatography materials which can be used in conducting gel filtration separations are readily available from several manufacturers such as Amersham Pharmacia Biotech, Bio-Rad, TosoHaas, or Whatman. This technology is well-known by those familiar with the art.
Gel filtration chromatography is typically carried out by a gel filtration matrix contained within a column. This column typically has vertical dimensions which are greater than its diameter. A mixture of molecular species is applied to this gel filtration matrix. The mixture, also known as a plug, then moves in a continuous stream of liquid which flows through the matrix. As the plug moves through the matrix, small molecules in the mixture diffuse into the pores of the matrix while large molecules do not. The particular gel filtration matrix that is chosen will determine the size of the molecules that will diffuse into it. As a result of this diffusion, larger molecules will emerge from the outlet of the column first, followed by the smaller molecules. The time between the emergence of the molecules is sufficient to allow the molecules to be separated based on their relative molecular sizes. A separation of this type may also be conducted in a batch mode, wherein several samples are introduced sequentially into the gel filtration device.
Gel filtration chromatography is effective for large sample volumes and is often used in industrial applications. However, it is difficult to process small samples (less than 1 mL) in this way. Small samples require the use of complicated and expensive equipment to detect the molecules of various sizes as they emerge from the column and to collect each small sample separately.
When the desired molecular species to be separated from a sample has a molecular weight greater than about 10,000 MW, and the sample size is about 1 mL to about 100 mL, then dialysis may be used to remove molecular species below about 10,000 MW. For smaller sample volumes, such as between about 100 xcexcL and 1 mL, dialysis devices may be used to remove these smaller species. This technique is sometimes called xe2x80x9cdesalting.xe2x80x9d However, this process requires several hours and the extent of removal of the small species may only reach 90-99%.
Accordingly, the above methods are not desirable when very small samples are being processed or where a very effective removal of small molecular weight molecules must be achieved. In these situation, single-sample spin columns filled with gel filtration material and designed to be used in microcentrifuges are used. These spin columns, available either hydrated or with a dehydrated dry mix gel filtration material, are available from several manufacturers. For example, CENTRI-SEP and CENTRI-SPIN spin columns are available from Princeton Separations. Additionally, MICROSPIN(trademark) columns are available from Pharmacia and SEQueaky KLeen columns are available from BioRad. These columns contain an amount of gel filtration material that, when swollen, allows recovery of high molecular weight molecules from samples of 20 xcexcL to 100 xcexcL in volume.
In operation, these hydrated spin columns are inserted into a centrifuge tube, known as a wash tube, and are centrifuged at a relative centrifugal force. The liquid outside the pores of the filtration matrix, known as interstitial fluid, is expelled from the spin column into the wash tube and is then discarded. The sample mixture is then added to the partially dried spin column and the spin column is inserted into a second centrifuge tube, known as a collection tube, and again centrifuged at a relative centrifugal force. This centrifugation causes small molecules in the sample to diffuse into the pores of the gel filtration material. The interstitial fluid is then expelled from the column and collected in the collection tube. The desired large molecular weight molecules are found in the expelled liquid in approximately the same volume as they existed in the original sample.
Gel filtration chromatography has many applications. This type of separation is useful in a number of assays, reaction cleanup steps, and analytical sample preparation. For instance, it may be used in the removal of non-incorporated radioactive nucleotides following an enzymatic labeling reaction of nucleic acid fragments, in the removal of non-incorporated fluorescent dyes following a protein labeling reaction, such as the labeling of an antibody, in the removal of salts and buffers from samples prior to analysis by capillary electrophoresis or mass spectrometry, or in the removal of non-incorporated labeled precursors from reactions prior to analysis of samples for automated DNA sequence determination.
Laboratories operating automated fluorescent DNA sequencing may process 20 to 2000 samples in one day. As such, single-sample spin columns are not suitable for the very high throughput labs at the high end of this range. The requirements of these laboratories are met by the availability of filter plates in the standard 96 well microtiter dish format. In this format, 96 individual wells are concentrically arranged in an 8 by 12 array. These wells are filled with a gel filtration material similar to that in the single spin columns. These filled 96 well filter plates are available from several manufacturers including Princeton Separations, Edge BioSystems, Sigma and BioRad. These filter plates are processed in a similar fashion as individual spin columns with the exception that these plates may accept samples as small as 5 xcexcL and require a special swing-out rotor to accommodate these 96 well plates in a bench-top centrifuge instead of the more common microcentrifuge. Of course, these plates are most efficient if 96 samples are being processed. Further, when these plates are used in applications requiring very efficient removal of small molecules ( greater than 99.99%) such as automated DNA sequencing, the techniques used in applying samples to the wells are extremely critical. Poor sample application technique can easily reduce the efficiency of removal to 90% or less.
As a result of the inability of current microcentrifugation techniques to process very small sample volumes and the inability of bench-top centrifugation techniques to process small numbers of samples and to easily remove small molecules with efficiency greater than 99.99%, a need exists for a device which will allow for very small sample volumes to be processed in a gel filtration technique while permitting a removal effectiveness greater than 99.99% and which can be operated in readily available equipment with little or no modification. The present invention is directed towards meeting these and other needs.